Vasoactive agents, such as thrombin and histamine, induce endothelial cell (EC) monolayer barrier dysfunction which is involved in a number of disease processes, including atherosclerosis. Monolayer barrier dysfunction is, at least, partially due to EC contraction which occurs via signaling events that culminate in myosin light chain (MLC20) phosphorylation. The phosphorylation of MLC20 by myosin light chain kinase (MLCK), is an obligatory step in contraction by smooth muscle and nonmuscle cells. Information is limited regarding events which regulate MLCK, a Ca2+/calmodulin (CaM)-dependent enzyme. However, it has been shown that thrombin and histamine induce the phosphorylation of MARCKS (myristoylated alanine-rich C kinase substrate), a CaM-binding protein. In the current proposal it is hypothesized that receptor agonist-induced MLCK activation in cultured EC is regulated by phosphorylation of MARCKS and CaM. To test this hypothesis the following Specific Aims (SA) are proposed: SA#1)To characterize agonist-induced MARCKS phosphorylation and MLCK activity in cultured EC. Upon phosphorylation by protein kinase C, MARCKS releases CaM that can then be used as a cofactor for MLCK. MARCKS phosphorylation will be determined and correlated with MLCK activation (as assessed by MLC20 phosphorylation) to evaluate whether the phosphorylation of MARCKS can be involved in agonist-induced MLCK activation. SA#2) To determine agonist-induced phosphorylation of CaM in cultured EC. CaM phosphorylated by either the insulin receptor or casein kinase II no longer augments, but inhibits in vitro MLCK activity, suggesting a novel mechanism of MLCK regulation. Recently, it has been discovered that CaM can also be phosphorylated by MLCK and that this phosphorylation temporally follows MLC20 phosphorylation. CaM phosphorylation will be assessed and correlated with MLCK activation. SA#3) To characterize CaM phosphorylation by MLCK. CaM will be phosphorylated by MLCK in vitro and the characteristics of this reaction will be determined by biochemical means. SA#4) To determine the consequences of CaM phosphorylation on CaM- MLCK interactions. The effect of phosphorylated CaM on MLCK activation will be evaluated by fluorometric and molecular biological techniques. These studies will demonstrate whether a temporal and vectoral relationship between CaM phosphorylation and MLCK activation exists and provide important insights in the mechanisms of EC contraction and barrier dysfunction.